Kurt Ballmer - Hofer, Paul Scherrer Institut, Biomolekulare Wissenschaften, Molekulare Zellbiologie

Das PSI ist eine
Forschungsanstalt des ETH
Bereichs und entstand 1988
aus dem Zusammenschluss
von EIR und SIN.
Kurt BallmerBallmer-Hofer, Paul Scherrer Institut,
Biomolekulare Wissenschaften, Molekulare Zellbiologie
52325232-Villigen
[email protected]
www.psi.ch
Das PSI betreibt
Grossanlagen wie die SLS
und die SINQ für die
Forschung, insbesondere für
die Materialforschung.
Das PSI betreibt
Protonentherapieanlagen
(OPTIS, PROSCAN) für
Tumorpatienten und eine
Abteilung für die
Herstellung von
Radiopharmazeutika für
Diagnostik und Therapie.
PAUL SCHERRER INSTITUT
Proton Therapy Program
The spot scanning technique for proton pencil beams –
physicists know how to deal with „their“ medium and
how to introduce new technologies into medical application
A thin proton beam (pencil beam)
is directed onto a target within
the human body.
The penetration is defined
by the energy of the beam and
the densities of the various
tissues which slow down the particles.
The beam, and thus the spots,
are magnetically deflected, i.e. are
scanned, according to the shape
of the target volume in the body.
The dose of each single spot
and of the entrance region
(plateau) are added up to the
prescribed dose for the whole
volume. The use of several
beam angles results in
excellent, 3-D conformation
of the high dose to the target
volume.
Gudrun Goitein
1
PAUL SCHERRER INSTITUT
Proton Therapy Program
The Spot Scanning Gantry at PSI
Gudrun Goitein
Die Rolle der Vaskularisierung beim Wachstum von Tumoren; Tumorwachstum benötigt
eine Versorgung mit Blut
Judah Folkman: Tumor angiogenesis, therapeutic implications
(1971)
2
Anti-Angiogenese; ein Konzept zur Eindämmung des Tumorwachstums, seine
Versprechungen und Erfolge
Tumorzellen überleben bevorzugt
entlang bestehender Blutgefässe.
Tumorzellen überleben bevorzugt entlang bestehender Blutgefässe
A. Frühe Tumoren wachsen entlang
bestehender Gefässe;
B. Die existierenden Gefässe bilden
sich zurück;
C. Regression des Tumors oder
Angiogenese.
Tumorzellen in rot
KB1
3
Slide 6
KB1
J. Holash et al. Science 284:1994 (1999)
Kurt Ballmer-Hofer; 01.11.2004
Der angiogene Schalter bestimmt, ob Blutgefässe überleben, neu gebildet
werden oder absterben
Zelluläre Faktoren, die die Angiogeneses regulieren
Pro-angiogene Faktoren
Anti-angiogene Faktoren
VEGF, Vascular endothelial growth factor
bFGF, basic fibroblast growth factor
PlGF, Placenta growth factor
PDGF, Platelet-derived growth factor
TGFα, Östrogene, EPO
EGF, IGF 1, IL-3, IL-8
Angiopoietins 1,2
Angiogenin
HIV-1 Tat protein, RGD-peptide
Tumor Necrosis Factor α (TNFα)
...
Thrombospondin (ECM-Protein)
Endostatin (collagen XVIII fragment)
Angiostatin (plasminogen kringle-domain)
Antibodies against VEGF/VEGFR or Integrins
Soluble VEGFR, ‚VEGF trap‘ (sFlt, Ig domain)
Inhibitors of VEGFR kinase, e.g. SU5416
Interferonα
TNFα
PEX, a noncatalytic fragment of MMP-2
Kininostatin, a kinin fragment Bradykinin
...
Tissue specific factors
EG-VEGF, endocrine gland derived VEGF
...
Tissue specific factors
Pigment epithelium derived factor, PEDF
...
4
KBH2
Tumoren wachsen besser in Anwesenheit von Angiogenese auslösenden Zellen
Ziele für anti-angiogene Therapie
5
Slide 9
KBH2
Engraftment of MOLT-3 cells is fostered by coadministration of Kaposi’s sarcoma cells.
(A) Macroscopic appearance of the tumors formed by MOLT-3 cells in NOD SCID mice.
(Left) Picture obtained 8 weeks after the bilateral s.c. injection of 5 106 MOLT-3 cells.
Although these cells failed to produce a visible mass in living mice, small tumors were
found at autopsy in 11 of 50 sites injected with MOLT-3 cells (22%); tumor volumes
ranged between
180 and 310mm3. (Right) Picture obtained 8 weeks after bilateral s.c. injection of 5
million MOLT-3 cells plus 5 million irradiated KS-IMM cells; tumors were found in 56 of
56 injection sites (100%), and their volumes ranged between 1,240 and 1,780mm3. (B)
Flow-cytometric analysis of in vitro-cultured MOLT-3
and KS-IMM cells (in vitro) or single-cell suspensions obtained through mechanical
dissociation of the tumors (ex vivo). Cells were incubated with a phycoerythrin-labeled
anti-human CD5 mAb and analyzed on an EPICS-Elite
cytofluorimeter. The percentage of positive cells is indicated. (C) Expression of
angiogenic factors and cytokines in MOLT-3 and KS-IMM cells. PCR products were
evaluated by electrophoresis on 1.5% agarose gels with ethidium bromide
staining. (D) Tumor growth curves of s.c. xenotransplants in NOD SCID mice. MOLT-3
cells were injected either alone or with other cell types. The tumor volume was plotted
as a function of time (weeks after transplantation). Groups (F) and (■) received MOLT-3
cells inoculated with equal numbers of irradiated KS-IMM cells or irradiated MOLT-3
cells, respectively; group (E) received MOLT-3 cells into one flank and irradiated KS-IMM
cells into the contralateral flank. In a group of mice (ƒ), irradiated KS-IMM cells were
injected 21 days after MOLT-3 cell injection. The tumor volumes of the experimental
group (F) evaluated after 9 weeks differed significantly compared with the (ƒ)
experimental group.
Indraccolo et al. PNAS 103 4216 (2006)
K. Ballmer-Hofer; 18.06.2007
Immunoliposomen für Tumortargeting
Rezeptor spezifische scFv
Antikörper, kovalent gebunden
Lipophile, membrangebundene
Toxine (5-FdU-NOAC, NOACETC, Epothilones)
Hydrophile, eingekapselte
Toxine (Mitoxantron, Peptide,
Proteine, antisense Oligos,
DNA, siRNA).
R. Schwendener
Phagendisplay-Technik für die Herstellung von scFv Antikörpern
Phagenreinigung
Positive Bakterien isolieren
und aufziehen
Infektion von
Bakterien mit Phagen
Gebundene Phagen
eluieren
Phagenbibliothek zu
Antigen zugeben
Ungebundene Phagen
entfernen
6
Tumor Targeting mit Immunoliposomen
1. Derivatisiertes Protein 2. Koppeln an Liposomen
O
+
3. Bindung an Zellen
O
N CH
C
2
O
O
O
C
N CH2
O
4. Akkumulation im Tumor und therapeutische Effekte
12.5
Controls untreated
1600
empty liposomes
scFv liposomes
% injected dose / g tumor
PEG-liposomes
1400
scFv-PEG-liposomes
Tumor growth (%)
10.0
7.5
5.0
2.5
0.0
Inhibition des
Tumorwachstums
50-60%.
scFv-dimers
5FdU-NOAC liposomes
1200
scFv-5FdU-NOAC-liposomes
1000
800
600
400
200
5
60
120
360
1440
Time (min)
0
0
1
2
3
4
5
6
7
8
Days
Treatments (i.v.)
C. Marty, R. Schwendener, D. Neri
Behandlung von F9 Teratocarcinomatumoren mit anti-ED-B scFv Liposomen
5-FdU-NOAC-Lipos
scFv-5-FdU-NOAC-Lipos
Control liposomes
Untreated
scFv dimers
scFv-Lipos
Treatment schedule: 5x every 24 h, tumors collected on day 8.
7
Die Rolle von Tumor assoziierten Makrophagen (TAMs) bei der Tumorprogression und
der Metastasenbildung
TAMs werden in der Umgebung des
Tumors konditioniert:
- um Angiogenese zu ermöglichen
- die extracelluläre Matrix abzubauen
- Tumorzellmotilität zu erhöhen
- sie präsentieren aber Antigen sehr schlecht
Tumorwachstum und
Metastasierung
Pollard, Nat. Rev.
Cancer 4, 2004
Hohe TAM Frequenz korreliert mit
schlechter Prognose
Inhibition der Angiogenese auf der Hühner Chorioallantois mit VEGF spezifischen scFv
Antikörpern
PBS
VEGF164
2.5 mm
S. Zeisberger
VEGF164 + scFv-A1
2.5 mm
0.1 mm
2.5 mm
VEGF164 + scFv-H9
2.5 mm
0.1 mm
8
Macrophage viability (%)
Bisphosphonate wie Chlodronat töten spezifisch Makrophagen ab
100
75
IC 50 = 2.8 mM = 1 mg/ml
50
PBS
Empty liposomes
25
Clodrolip
0
w/o
0.001
0.01
0.1
1
10
Clodrolip (mg/ml)
C ell density [% ]
125
MΦs
HUVECs
F9
A673
100
75
*
50
*
25
0
w/o
Empty liposomes
Free clodronate
Clodrolip
Incubation (1 mg/ml, 6h, 37°C, 5% CO2)
S. Zeisberger, C. Marty, R. Schwendener
Depletion von Immunzellen in der Milz
Macrophages
ER-TR 9
F4/80
Dendritic cells
CD68
FDC
CD11b
CD11c
B-cells
T-cells
B220
CD3
Clodrolip
Free
clodronate
PBS
MOMA1
9
Depletion von CD11c+ Tumor assoziierten dendritischen Zellen (TADC)
scFv-A1
scFv-H9
CL
CL/scFv-H9
day 22
CD11c
day 16
PBS
TAM Depletion mit Chlodronat und VEGF Antikörpern
Reduction of F 4/80 positive TAMs
scFv-A1
scFv-H9
Clodrolip
CL/scFv-H9
Day 22
Day 16
PBS
TAMs, day 22 (%)
125
100
75
50
25
S. Zeisberger, R. Schwendener
***
0
PBS
(ScFv')2-A1
(ScFv')2-SZH9
Clodrolip
***
Clodrolip + (scFv')2-SZH9
Treatment
10
Zerstörung der Tumorvaskulatur mit Chlodronat und VEGF Antikörpern
scFv-A1
scFv-H9
Clodrolip
CL/scF-H9
Vessel density, day 22 (%)
Day 22
Day 16
PBS
S. Zeisberger, R. Schwendener
100
75
*
50
25
***
***
0
PBS
(ScFv')2-A1
(ScFv')2-SZH9
Clodronate
Clodronate + (scFv')2-SZH9
Treatment
Anti-angiogene Kombinationstumortherapie im Mausmodell
700
Humane A673 Rhabdomyosarcomatumoren in CD1 Nacktmäusen
ScFvSZ H9 + CL
300
200
100
0
(C) 1
2
3
4
(C+S) 5
(S) 6
(S) 7
(S) 8
(C+S) 9
(S) 10
(S) 11
(S) 12
(C+S) 13
14
15
16
17
18
19
20
21
22
0
Days after A673-tumor injection and treatment scheme
0.6
0.4
0.2
0.0
ScFvSZH9 + CL
CL
400
0.8
CL
ScFvSZ H9
Controls
Tumor growth (%)
ScFv A1
500
ScFvSZH9
PBS
Tumor volume, day 16, x 1000 (mm3)
1.0
600
S. Zeisberger, C. Marty, R. Schwendener
11
Korrelation der TAM Depletion mit der Gefässdichte
Kontrolle
800
F4/80+ macrophages
Makrophagendichte
700
MOMA1+ macrophages
600
500
r = 0.755, P < 0.0001
400
300
200
100
0
r = 0.735, P < 0.0001
0
100
200
300
400
Vessel density (CD31)
Behandelt
Zusammenfassung
¾
Die Herstellung rekombinanter Proteine, wie z.B. Antikörper, ermöglicht die Einführung
neuer Therapien bei der Tumorbekämpfung. Als Ziele für die Behandlung mit
rekombinanten Molekülen eignen sich:
• tumorspezifische Oberflächenrezeptoren
• Moleküle, die auf dem Tumorstroma oder der Tumorvaskulatur exprimiert werden
• lösliche Wachstumsfaktoren, die direkt das Tumorwachstum begünstigen
• lösliche Wachstumsfaktoren, die bei der Rekrutierung von Zellen, die das
Tumorwachstum begünstigen, eine Rolle spielen (z.B. Zellen des Immunsystems).
¾
Anti-angiogene Therapie kann komplementär zu klassischen Behandlungen eingesetzt
werden, wird als Einzeltherapie aber kaum langfristig wirksam sein (Resistenz durch
Wechsel der angiogenen Faktoren).
¾
Liposomen sind ideale ‘Delivery Vehicles’ für zielgerichtete Therapien gegen Tumorzellen,
müssen aber durch Modifikation der Oberfläche den Bedingungen im Organismus und im
Zielgewebe angepasst werden.
¾
Bisphosphonate können als zusätzliche Therapeutika die Wirksamkeit anti-angiogener
Therapien drastisch erhöhen. Sie richten sich vor allem gegen TAMs.
12
Aber, so einfach ist die Geschichte nun doch nicht
KBH4
Regression der Vaskulatur nach anti-angiogener Behandlung
T Kamba and DM McDonald
British Journal of Cancer (2007)
96, 1788 – 1795
Mechanisms of adverse effects
of anti-VEGF therapy for cancer
13
Slide 26
KBH4
Regression of capillaries in vasculature of normal adult mice after inhibition of VEGF
signalling. (A–D) Confocal microscopic images showing
capillaries in pancreatic islets (A and B) and villi of small intestine (C and D) under
baseline conditions and after VEGF inhibition. After Ad-sVEGFR-1 for 14
days, endothelial cells of some capillaries have regressed, leaving pericytes (red, NG2,
arrowheads) at sites of regression (Kamba et al, 2006). (E) Comparison of
VEGFR-2 and VEGFR-3 immunofluorescence in pancreatic islet capillaries after VEGF
inhibition. Stronger endothelial cell VEGFR-2 immunoreactivity under
baseline conditions (upper left) than after Ad-sVEGFR-1 for 14 days (upper right).
Stronger endothelial cell VEGFR-3 immunoreactivity under baseline
conditions (lower left) than after Ad-sVEGFR-1 for 14 days (lower right). (F) Bar graphs
showing fluorescence intensities of VEGFR-2 and VEGFR-3
immunoreactivities under baseline conditions and after Ad-sVEGFR-1 for 14 days (Kamba
et al, 2006). *Po0.05, significantly different from corresponding
control. wPo0.05, significantly different from islets. (G–I) Fluorescence micrographs of
thyroid capillaries stained for CD31 immunoreactivity show dense
vascularity under baseline conditions (G), loss of half of the capillaries after AG-013736
for 7 days (H), and complete regrowth of vasculature during 14 days
after end of treatment (I) (Kamba et al, 2006). Scale bar in I: 25 mm in (A–D); 40 mm
in (E) and (F); 160 mm in (G–I).
Kurt Ballmer-Hofer; 14.08.2008
Rückgang der Fenestrierung inKBH3
den Nierenglomeruli
Einige Nebenwirkungen von anti-VEGF Therapien
¾ Bluthochdruck (Verlust von NO signaling)
¾ Proteinuria (Verlust der Fenestrierung der EZ und von Podocyten,
Verlust der Filtrierbarriere)
¾ Reduktion der Wundheilung (bei Operationen)
¾ Gastrointestinale Komplikationen (Darmperforationen, Defekte in den
Kryptzellen)
¾ Blutungen, Thrombosen (NO, PG Reduktion, Epo Ueberproduktion)
¾ Störungen der Blut-Hirnschranke
¾ Herzinsuffizienzen
¾ Endokrine Probleme
14
Slide 27
KBH3
Reduction in endothelial fenestrations (arrowheads) after inhibition of VEGF signalling.
(A and B) Transmission electron microscopic images of
islet capillaries showing thin endothelium and abundant fenestrations with diaphragms
under baseline conditions compared to thick endothelium, few
fenestrations, and abundant caveolae after AG-013736 for 21 days (Kamba et al, 2006).
(C and D) Transmission EM images of renal glomerular capillaries
comparing thin endothelium and abundant fenestrations under baseline conditions with
thick endothelium and few fenestrations after Ad-sVEGFR-1 for 14
days (Kamba et al, 2006). (E and F) Scanning electron microscopic images of luminal
surface of glomerular capillaries showing abundant endothelial
fenestrations under baseline conditions and few fenestrations after Ad-sVEGFR-1 for 14
days (Kamba et al, 2006). (G) Bar graph showing significantly higher
concentration of TSH in serum as a measure of altered thyroid function after AG-013736
for 21 days. (H) Bar graph showing increasing amount of
proteinuria, indicated by proportion of mice with Albustix values of þ þ or greater (X100
mg albumin/dl of urine), with increasing dose of AG-013736 for
7 days. (I) Diagram of hypothetical shuttling of diaphragms between endothelial
fenestrations and caveolae, with VEGF inhibition driving the process to the
right and VEGF signalling driving it to the left (Kamba et al, 2006). Scale bars: 0.3 mm in
(A) and (B); 1 mm in (C) and (D); 0.5 mm in (E) and (F).
Kurt Ballmer-Hofer; 14.08.2008
Zitate des Experten (McDonald et al.)
¾
¾
¾
¾
… a more complete understanding of the basic biology of tumor vessels will be necessary to
fully appreciate the consequences of vessel leakiness in cancer. New research tools such as
intravital measurements of tumor blood flow and vessel leakiness, in vivo phage display,
magnetic resonance imaging, and use of selective angiogenesis inhibitors will contribute to
this understanding (2002).
In … tumors, pericytes did not degenerate to the same extent as endothelial cells, and those
on surviving tumor vessels acquired a more normal phenotype. Vascular basement membrane
persisted after endothelial cells degenerated, providing a ghost-like record of pretreatment
vessel number and location and a potential scaffold for vessel regrowth. The potent antivascular action observed is evidence that VEGF signaling inhibitors do more than stop
angiogenesis. Early loss of endothelial fenestrations in RIP-Tag2 tumors is a clue that vessel
phenotype may be predictive of exceptional sensitivity to these inhibitors (2004).
VEGF inhibitor … effects are typically downstream consequences of suppression of cellular
signalling pathways important in the regulation and maintenance of the microvasculature.
Downregulation of these pathways in normal organs can lead to vascular disturbances and
even regression of blood vessels, which could be intensified by concurrent pathological
conditions. These changes are generally manageable and pose less risk than the tumours
being treated, but they highlight the properties shared by tumour vessels and the
vasculature of normal organs (2007).
The MR imaging-assayed acute change in vascular leakiness after a single dose of
bevacizumab was an early, measurable predictive biomarker of tumor angiogenesis treatment
response (2008).
Tumor targeting
Reto Schwendener (IMCR UNIZH)
Steffen Zeisberger
Conni Marty
Anne Fjällman
http://mcb.web.psi.ch
[email protected]
Funding
Swiss National Science Foundation, Novartis Foundation, Sassella
Stiftung, Oncosuisse, Paul Scherrer Institut
Collaborators at Structural Biology and SLS at PSI
Rolf Jaussi, Andrea Prota, Jack Missimer, Dirk Kostrewa, Michel Steinmetz, Fritz Winkler, Clemens
Schulze-Briese, Armin Wagner, Franz Pfeiffer, Oliver Bunk
Outside collaborators
Giorgios Skiniotis & Tom Walz, Harvard Medical School; Jeannette Wood, Novartis Pharma; Jody Haigh, VIB
Gent; Functional Genomics Center University of ZH, Lena Claesson-Welsh, Uppsala University
15
KBH3
Notch Signalübertragung antagonisiert VEGF und stimuliert das Tumorwachstum
KBH1
Metabolic control of cancer cell growth
16
Slide 31
KBH3
VEGF and Dll4 regulate tumor angiogenesis. VEGF activates the VEGFR-2 signaling
pathway
to stimulate tumor angiogenesis. Dll4 binding to Notch negatively regulates sprouting
and branching during tumor angiogenesis resulting in a functional vascular network.
Anti-VEGF treatment inhibits angiogenesis and suppresses tumor growth. In contrast,
Dll4 blockade stimulates nonproductive tumor vascularization, resulting in inhibition of
tumor growth.
http://www.nature.com/naturebiotechnology
Hicklin D.J. Nature biotech 25 2007
K. Ballmer-Hofer; 22.06.2007
Slide 32
KBH1
The metabolism of glucose and its regulation by oncogenic signals. (A) Glycolysis breaks
down glucose in the cytoplasm to
generate pyruvate. In the absence of oxygen, pyruvate is reduced to lactate. In the
presence of oxygen, pyruvate is oxidized by PDH
to acetyl-CoA, which enters the mitochondria and the TCA cycle. Oxidative
phosphorylation generates ATP as an energy source and
also produces ROS. ROS can cause mitochondrial damage and apoptosis, both by
stimulating the release of apoptotic factors and
through activation of Kv1.5 channels. Glycolytic metabolism can be promoted by
oncogenic changes. For instance, the tumor suppressor
p53 inhibits glycolysis through TIGAR induction and stimulates oxidative phosphorylation
through SCO2 induction. In cancer
cells with mutated p53, ROS-induced apoptosis is thus decreased. The oncogene DJ-1
also blocks ROS-induced apoptosis and may
increase glycolysis by inhibiting PTEN and thereby activating PI-3′ kinase and PKB.
Hypoxia can induce glycolysis in cancer cells
through HIF-1α stabilization and activation, and thereby the up-regulation of glycolytic
enzymes. Furthermore, hypoxia suppresses oxidative
phosphorylation through the transcriptional activation of the gene encoding PDK. By
inhibiting PDK, DCA may shift cancer cell
metabolism back toward oxidative phosphorylation. Some cancer cells may use
nonglucose energy sources such as amino acids, fatty
acids, or nucleic acids. How p53 and hypoxia influence the metabolism of these energy
sources is unknown. (B) Wild-type p53 and
DCA shift cancer cells toward oxidative phosphorylation and apoptosis. Oncogenic
changes and hypoxia shift cancer cells toward glycolytic
metabolism and survival.
Pan and Mak, STKE 381 e14 2007
K. Ballmer-Hofer; 27.06.2008
Metabolic control of cancer cell growth
17
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